Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
A variety of different cell types are currently used to express proteins that are commercially relevant in a range of diagnostic, prophylactic, therapeutic and/or research applications. Currently, the production of such proteins is routinely carried out in cells such as bacteria, yeast, fungi, insect and non-human mammalian cells.
Cells frequently modify proteins with a multitude of post-translational modifications including, but not limited to, glycosylation, acylation, phosphorylation, methylation, sulfation, prenylation and lipidation. These modifications are species specific and, as such, the cells currently used in the production of commercially relevant proteins exhibit post-translational modifications that are distinct from the post-translational modifications observed on proteins expressed from human cells or occurring naturally in the human body. For example many non-mammalian cell types used to produce commercially relevant proteins either lack the capacity to glycosylate proteins or exhibit glycosylation patterns that are different to the glycosylation patterns exhibited by proteins expressed in human cells.
Even in non-human mammalian expression systems such as Chinese hamster ovary (CHO) cells, significant differences in the glycosylation patterns are documented compared with that of human cells. For example, CHO cell lines used for recombinant protein expression lack a functional (α 2, 6) sialyltransferase enzyme for synthesis of (α 2, 6)-linked terminal sialic acids which are present in human cells. Furthermore, the sialic acid motifs that are present on CHO-cell-expressed glycoproteins are prone to degradation by a CHO cell endogenous sialidase (Gramer et al. Biotechnology 13 (7):692-&, 1995).
As a result of the distinct post-translational modification repertoires of non-human expression systems, proteins expressed from them may exhibit physiochemical and pharmacological characteristics such as half-life, immunogenicity, stability and functional efficacy that are distinct from human cell-derived proteins. This can substantially impact on the clinical utility of these proteins.
There is also growing evidence that in addition to its species-dependent nature, post-translational modifications can also be tissue- and even cell type specific within the same species. This is particularly relevant to tissue- and cell type-specific expression of proteins exhibiting terminal glycosylation (Feizi Nature 314: 53-54, 1985; Rademacher et al Annu Rev Biochem 57: 785-838, 1988). Specifically, it has been shown that three sialyltransferases, which attach terminal sialic acids to glycoprotein sugar chains, exhibit striking differential expression in seven tissues of the rat (Paulson et al J. Biol. Chem. 264: 10931-10934, 1989). This provides support for tissue specific glycosylation of the same protein. Furthermore, studies with two isoforms of a highly phosphorylated glycoprotein (mouse osteopontin) expressed by mouse fibroblasts and mouse osteoblasts from bone marrow exhibited major differences in their degree of phosphorylation, which correlated with differences in biological activity. These results indicate that the function of osteopontin produced by different cell types is distinct (Christensen et al J. Biol. Chem. 282(27): 19463-19472).
Efficacious cell systems suitable for the production of biologics exhibiting fully human characteristics ideally should satisfy a number of criteria, including but not limited to:                a) derivation from human tissue;        b) high density growth in culture;        c) exhibit commercially viable protein yields;        d) allow stable foreign gene introduction;        e) permit gene amplification methods;        f) allow use of a parent cell for monoclonal antibody production as in human-human hybridomas;        g) exhibit stable long-term protein expression;        h) the ability to grow in a serum-free and glutamine-free medium;        i) lack endo-peptidase activity, thus reducing protein degradation,        j) be free of pathogenic agents including viral DNA and mycoplasma;        k) produce proteins that exhibit post-translational modifications that are functionally similar to, or the same as post-translational modifications that occur on naturally-occurring human proteins, preferably tissue and cell specific. These post-translational modifications may include, but are not limited to, carbohydrate moieties on glycoproteins.        
Whilst a number of human host cells or heterohybridomas exist for the expression of human protein, none of them successfully satisfy all of the criteria listed above. Most notably, attempts to express and isolate proteins from existing human cell expression systems in clinically useful yields have resulted in limited success.
Protein expression in eukaryotic cells is controlled at multiple stages, including: (a) the influence of regulatory factors on the genes in the chromatin; (b) regulation of initiation of transcription; and (c) post-translational modification. These different stages are thought to be developmental stage- and/or tissue-specific. Thus, when an exogenous gene encoding a desired protein is incorporated into a cell, expression of the desired protein may be less than optimal. Problems such as lack of stable expression (Li et al., Proc Natl Acad Sci USA 95: 3650-3654, 1998; Miyaji et al., Cytotechnology, 3: 133-140, 1990; Miyaji et al., Cytotechnology 4: 173-180, 1990; Miyaji et al., Cytotechnology 4: 39-43, 1990; Satoh et al., Cytotechnology 13: 79-88, 1993), low expression yields (Airoldi et al, Cancer Research 61:1285-1290, 2001; Hosoi et al. Cytotechnology 7: 25-32, 1991) and non-optimal post-translational modifications (Shinkawa et al., J. Biol. Chem. 278:3466-3473, 2003) may result. All of these factors may influence the potential commercial utility of the protein.
As one example, a subline of Namalwa cells (human B lymphoblastoid cells grown in suspension cultures and adapted to a serum and albumin-free medium), Namalwa KJM-1, was used for large scale production of alpha-interferon, which is an endogenous protein to Burkitt's lymphoma cells. However, when G-CSF protein foreign to Burkitt's lymphoma cells but endogenous for B cells (Airoldi et al, Cancer Research 61:1285-1290, 2001) was used as targeted protein for transfection via electroporation, the levels of G-CSF expression varied among multiple methotrexate (MTX) resistant clones and the highest G-CSF-producer clone had a specific productivity of only 2.4 μg/ml/day when adapted to serum free conditions.
Further, the specific productivity was depressed at high density culture when the cell number was above 7×105 cells/ml (Hosoi et al. Cytotechnology 7: 25-32, 1991). Even though the reported maximum G-CSF concentration was markedly improved and reached 41 μg/ml, in order to achieve this, it required extensive and laborious manipulation of cell culture conditions with very tight control of pH. It also showed that the medium used for the optimal growth was different from that used for the optimal production, thus creating significant conflict between desired high density and high production rate, and resulting in an industrially non-viable system.
Because gene expression in eukaryotes is controlled in multiple steps, which include: (a) availability and accessibility of regulatory factors to the genes in the chromatin; (b) modulation on accessible promoters of the rate of specific initiation of transcription; and (c) subsequently post-transcriptional events at various steps, the presence of tissue specific and development specific transcription factors has a great influence on the expression of genes. Further, the gene regulation of a specific cell type requires cooperation of several cis-acting DNA regulatory sequences, which are binding sites for proteins that transmit molecular signals to genes (Blackwood et al., Science 281: 60-63, 1998). These sequences bind regulatory proteins to form complexes known as enhanceosomes (Marika et al., Curr Opin Genet Dev 11(2): 205-208, 2001). Thus, the further the targeted gene is away from its usual cellular environment when introduced into the human lineage specific host cells, stable expression and production of desired protein at high production levels are reduced. When Namalwa KJM-1 cells were transfected with genes of foreign proteins further away from being lineage specific proteins for lymphoblastoid cell lines such as beta interferon (Miyaji et al., Cytotechnology, 3: 133-140, 1990; Miyaji et al., Cytotechnology 4: 173-180, 1990) or human lymphotoxin (Miyaji et al., Cytotechnology 4: 39-43, 1990) or pro-urokinase (Satoh et al., Cytotechnology 13: 79-88, 1993), the transfection rate and the cell productivity were found to be even lower.
Efficient expression of foreign genes in human lineage specific cell lines also requires a careful, and sometimes, tedious selection of a suitable enhancer/promoter which would contain binding sites for nuclear factors available from human host cells. Finding such an enhancer/promoter might still result in limited suitability of such a promoter. For example, when several enhancers/promoters such as the simian virus 40 (SV40) early gene promoter, human cytomegalovirus (hCMV) major immediate-early gene promoter, Moloney murine leukaemia virus (Mo-MuLV) promoter, Rous sarcoma virus (RSV) promoter and chicken β-actin promoter, were investigated for more efficient expression of a foreign gene in Namalwa KJM-1 cells, the Mo-MuLV promoter was found to be about 10 times stronger than traditional SV40 earlier promoter and the high producer clones reached productivity of 30-40 μg/106 cells/day (Satoh et al., Cytotechnology 18:162-172, 1996). However, the problem with using retroviral vectors such as Mo-MuLV is that it is difficult to use for transfection of genes with inverting sequences (introns) because of their removal by the nuclear splicing machinery (Li et al., Proc Ntl Acad Sci USA 95: 3650-3654, 1998).
The mismatch between cellular and nuclear environment is augmented even further in the case of the proteins encoded by two genes such as antibodies. Further, whilst Namalwa KJM-1 cells were used for generation of human-human hybridomas, the antibody yields made this cell line unsuitable for industrial production.
As another example, human embryonic kidney cell line 293 has proven to be very easily transfected with genes of foreign origins with a high degree of stability. However, proteins derived from 293 transfectants have limited use and are usually suited for research purposes only because 293 cells include human adenovirus Ad5 DNA (HEK 293 cells). However, the greatest limitation in using the 293 cells in a commercial setting is its adherent nature. A number of attempts have been made to adapt 293 cells for efficient transfection in suspension using cost effective vehicles such as polyethyleneimine (Durocher et al., Nucleic Acids Res 30(2):e9, 2002; Schlaeger et al., Cytotechnology 30:71-83, 1999) or calcium phosphate (Girard et al, Cytotechnology 38:15-21, 2002; Jordan et al., Cytotechnology 26:39-47, 1998; Meissner et al., Biotechnol Bioeng 75(2):197-203, 2001). However, these vehicles result only in transient expression of recombinant proteins meaning that the transfection has to be repeated for each new batch of seeded culture. In order to achieve suspension growth and higher protein expression when EBV's oriP is present in the vector backbone, the 293 cells had to be genetically modified to stably express the Epstein Barr virus EBNA1 protein (293E) (Durocher et al., Nucleic Acids Res 30(2):e9, 2002; Parham et al., Cytotechnology 35:181-187, 2001; Schlaeger et al., Cytotechnology 30:71-83, 1999). Even after the transfection with EBNA1, the 293E cells when grown in serum free medium (HEK293 EBNA1), (prerequisite for large scale production) exhibit a very poor transfection rate most likely due to the presence of polyanions (heparin, dextran sulphate) that are added to prevent cell aggregation. Attempts have been made to mitigate this problem by supplementing medium with peptones obtained from enzymatic hydrolysis of animal sources such as meat, gelatin and casein (Pham et al., Biotechnol Bioeng 84(3):332-42, 2003). When HEK293 EBNA1 cell line was used for the production of Tie-2 (receptor tyrosine kinase for angiopoietin growth factors) and Neuropilin-1 ED (receptor that mediates neuronal cell guidance) the protein expression was limited by the low cell density cultures obtained when compared to those obtained in untransfected cultures. Also, the purity of >95% of resulting protein is suitable for research grade products only. In addition, the HEK293 EBNA1 cells are not suitable for production of monoclonal antibodies (mAbs).
Current strategies for production of therapeutic mAbs include the use of mammalian cell systems (i.e. CHO or NS0 transfectomas) to recombinantly produce mAbs derived from immunization of transgenic mice bearing human Ig genes (xenomice), humanization of rodent mAbs, or through screening of human mAb libraries (van Dijk et al., Curr. Opin. Chem. Biol. 5:368-374, 2001). Whilst in terms of their sequence, therapeutic mAbs have recently evolved into chimeric (rodent variable and human constant regions), humanized (human sequence except for rodent complementary-determining regions), and fully human antibodies (human Abs) to minimise the allergic response, the important aspect of a therapeutic mAb is its ability to elicit immune effector functions, such as antibody-dependent cellular cytotoxicity which is compromised if mAb is produced in non-human host cells that alter its native glycosylation pattern (Shinkawa et al., J. Biol. Chem. 278:3466-3473, 2003). In view of these facts, an ideal scenario is one where therapeutic antibodies are produced by human cells. In this case, fully human mAbs would be able to exert human effector functions and have very limited immunogenicity because of their native human structure.
The generation of hybridomas or Epstein-Barr virus (EBV)-transformed lymphoblastoid lines derived from human B cells has been reported (Kirman et al., Hybrid. Hybridomics 21: 405-414, 2002; Boerner et al., J. Immunol. 147: 86-95, 1991; Zafiropoulos et al., J. Immunol. Methods 200: 181-190, 1997). However, there is limited information on the characterization of these mAbs and the lines with respect to their long-term stability and suitability to manufacturing processes, especially the production levels and stability of Ig secretion during the entire batch manufacturing. Whilst cell lines producing human mAbs against human GM-CSF at cumulative titre of 1.2 g/litre during a 4-day run have been reported, these cell lines were derived from somatic cell hybridisation (fusion) of primary human B cells with heteromyelolymphoma K6H6/B5 cells (i.e. a mouse-human cell line obtained from a hybridisation of a human B cell lymphoma and a mouse myeloma cell (Li et al., Proc Natl Acad Sci USA 103(10):3557-3562, 2006).
In the instance of EBV-transformation, the difficulty has been the establishment of a completely immortalized human B cell line while maintaining stable antibody production. This is due to low efficacy of immortalization, the arrest of cell growth, and the dominant immortalization of IgM producing cells. Additionally, recent reports have shown that most EBV-transformed B cells have shortened telomeres and a limited life span, mostly before 160 population doubling levels (Sugimoto et al., J Virol 73:9690-9691, 1999; Toda et al., J Chromatogr B Analyt. Technol. Biomed. Life Sci. 787:197-206, 2003). In order to overcome this problem, attempts have been made to hybridise (or fuse) EBV-transformed B cells with a suitable partner cell line (expression system) but in effect these partner cell lines represented various combination of heterohybrids such as a trioma derived from a mouse-human heterohybridoma with a human B cell (Ainai et al., Hum Antibodies 15:139-154, 2006; Kalantarov et al., Hum Antibodies 11: 85-96, 2002; Karpas et. al., Proc Natl Acad Sci USA 98:1799-1804, 2001). When such a trioma was fused with primary EBV-transformed B cells producing an antibody to tetanus toxin (TT), it resulted in a tetroma having a quarter of the mouse component. Although stable production of mAbs to TT was possible after three-time consecutive cloning of tetromas, such repeated cell cloning steps are laborious and time-consuming. In addition, although the quantities of the mAbs produced by tetromas were sufficient for experimental purposes, the levels were insufficient for large-scale production of mAbs as pharmaceutical agents. It is still questionable whether immortalization in the presence of polyclonal activator CpG 2006 or co-ligation of CD19 or BCR might result in a complete system for an efficient production of specific mAbs in appropriate volumes for therapeutic use (Hartman et al., J Immunol 164: 944-953, 2000; Hur et al., Cell Prolif 38: 35-45, 2005; Traggiai et al., Nat Med 10: 871-875, 2004).
A number of approaches have been tried to use human cells for the production of biological substances such as growth factors, antibodies and soluble proteins.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.